JP2002516089A - Improved methods and materials for transformation - Google Patents

Abstract

Summary [57] Disclosed herein are novel methods and materials directed to transforming host cells and expressing exogenous RNA therein. In particular, a DNA-launching platform used to introduce a replicating viral segment attached to an exogenous polynucleotide into a cell, whereby the exogenous polynucleotide is expressed in the cell and confers a detectable trait. ) Is disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This invention was made with United States Government support awarded by the following agency: NIH
Grant number: GM35072. The United States Government has certain rights in the invention.

BACKGROUND RNA virus of the invention is known to be a valuable tool in the transformation of phenotypic and genotypic cell and tissue targeting. For example, a novel viral RNA
See US Patent No. 5,500,360 which discloses an expression vector. RNA in the genome of an RNA virus can be modified to include an exogenous RNA segment, and the modified RNA is introduced into a host cell and allowed to replicate therein, thereby expressing the exogenous RNA segment It has been shown that it can.

[0003] Current methods of inoculating a host cell with a modified RNA virus involve performing in vitro transcription of a particular strand and then introducing the resulting RNA transcript into the host cell. One problem with current inoculation methods is that RNA is rapidly degraded, which reduces infection efficiency. In addition, preparation of in vitro RNA transcripts is expensive and time consuming.

[0004] In addition, with the advent of plant transformation and genetic engineering, there has been much concern about the potential risk of disseminating dangerous traits into the environment. For example, genes that increase the stress and / or herbicide resistance of agriculturally important crops can be added to the surrounding less-desired, less-damaged plants by, for example, pollen, mechanical spraying, or insect spraying. "Leakage" may occur. This phenomenon can give rise to new “super weed” species, which can cause havoc in agriculture. Existing RNA virus-based vectors can be spread to non-target plants by mechanical means and / or insects. Such dissemination can be prevented by using a vector that can replicate and / or move only in the target plant that expresses the appropriate trans-acting factor. Thus, there remains a need for cheaper and more efficient methods for transforming target cells and tissues. In addition, there is a need for new transformation methods that reduce the potential risks associated with the undesired distribution of genetically engineered traits into the environment.

[0005] BRIEF DESCRIPTION OF THE INVENTION The present invention comprises the step of transfecting DNA delivery platform (DNA-launching platform) to the cells, the host cells for improved materials and methods for transformation. One aspect of the invention is that the improved viral RNA molecule is effective in a host cell so that the DNA delivery platform can be introduced into a host cell, in which the DNA delivery platform is replicated and expressed. "
It relates to a DNA delivery platform that encodes a modified viral RNA molecule downstream of a DNA-dependent RNA polymerase (pol) promoter, which can be “launching”. "Modified viral RNA molecule" as used herein
The term refers to a viral RNA that has changed from its natural state. Examples of changes in the viral RNA include, but are not limited to, removal of a portion of the viral RNA genome, insertion or replacement of exogenous RNA, and the like. An exogenous RNA segment can be present in a region of a viral RNA molecule such that it does not interfere with RNA replication. The art of such operations has been known to those skilled in the art for many years. Preferably,
The modified viral RNA molecule further comprises a ribozyme located proximal to the 3 'end of the modified viral RNA molecule. Viral segments may be capable of replicating in the presence or absence of trans-acting viral replication elements.

[0006] Another aspect of the invention relates to the use of a DNA delivery platform encoding a viral RNA molecule and an exogenous RNA segment, wherein the DNA delivery platform encodes the viral RNA segment or the exogenous RNA segment.
Introducing the RNA segment into a site that does not interfere with replication, thereby
A method for altering the genotype or phenotype of a host, wherein the exogenous RNA segment confers a detectable trait in the host cell. The invention applies to a variety of plant cells.

[0007] Yet a further aspect of the invention relates to a cell into which the DNA delivery platform of the invention has been introduced.

[0008] Yet another aspect of the invention relates to a plant comprising cells transfected with a DNA delivery platform.

[0009] The novel methods and materials of the present invention show that the use of the DNA delivery platforms of the present invention makes them more resistant to degradation of RNA inoculum, and that each DNA delivery platform provides a large number of RNA Provides greater inoculation efficiency of RNA virus to produce transcripts. Since the DNA delivery platform provides a genetically stable in-plant storage copy of the desired vector construct, sustained transcription of the DNA delivery platform will likely result in repeated re-inoculation of the host cell with the desired construct. It is. This helps to overcome the problem of gene instability, in which the expression of some genes from vectors based on plant and animal RNA viruses has been inhibited. Furthermore, the inoculation method of the present invention provides a much simpler means of producing the inoculum as a bulk used on a large scale, which is cheaper and more efficient than inoculating in vitro RNA transcripts.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1: pB1LR2-partial nucleotide sequence contains the BMV1a expression cassette.

SEQ ID NO: 2: pB1LR3-partial nucleotide sequence contains the BMV1a expression cassette.

SEQ ID NO: 3: pB2LR4--Partial nucleotide sequence contains the BMV 2a expression cassette.

SEQ ID NO: 4: pB2LR5--Partial nucleotide sequence contains the BMV 2a expression cassette.

SEQ ID NO: 5: pB12LR6-partial nucleotide sequence includes BMV 1a and 2a expression cassettes.

SEQ ID NO: 6: pB12LR7-partial nucleotide sequence includes BMV 1a and 2a expression cassettes.

SEQ ID NO: 7: pB12LR8-partial nucleotide sequence includes BMV 1a and 2a expression cassettes.

SEQ ID NO: 8: pB12LR9-partial nucleotide sequence includes BMV 1a and 2a expression cassettes.

DETAILED DESCRIPTION OF THE INVENTION In order to facilitate understanding of the present invention, certain terms are defined herein throughout this specification. The term "RNA virus" as used herein,
A virus whose virus genome is double-stranded or single-stranded RNA and whose single strand is a (+) strand or a (-) strand.

As used herein, the term “transfection” or “transfected” refers to by mechanical inoculation, electroporation, agroinfection, particle-bombardment, microinjection, or other known methods. Introducing exogenous DNA or RNA into cells.

“Transformation” or “transformed” as used herein refers to the stable integration of exogenous DNA or RNA into a cell that causes a permanent genetic change in the cell. Thus, one of skill in the art would understand that transfection of a cell may result in transformation of the cell.

As used herein, “launched” refers to a preferably transcribed, preferably replicated, polynucleotide transcribed from a DNA delivery platform as described herein.

As used herein, the term “cis-acting element” refers to an RNA that must be present in cis, ie, must be present as part of the respective viral strand as a requirement for replication of that strand. Shows a portion of the viral RNA genome. Viral replication appears to depend on the presence of one or more trans (diffusible) elements that interact with cis-acting elements to effect RNA replication. If trans-acting elements are required for replication, they are provided and need not be present on the modified viral RNA, or need not be encoded on the RNA, but may be present in the infected cell by some other means. You may make it available. For example, the trans-acting replication function may be provided by other unmodified or modified components of the viral genome transfected into the cell simultaneously with the modified RNA. The same approach can be used for other trans-acting functions, including motor proteins, coat proteins, and other functions. The target cell may also be pre-modified, for example, the cell may have been previously transformed from a chromosome to provide constitutive expression of a trans-acting function. A cis-acting element is comprised of one or more segments of viral RNA that must be present on an RNA molecule to be replicated in a host cell by RNA replication. The segments are most likely the 5 'and 3' end portions of the viral RNA molecule and may include other portions and / or the viral open reading frame as well.
Thus, a cis-acting element is defined in functional terms: any modification that disrupts the ability of an RNA to replicate in a cell known to contain an essential trans-acting element appears to be a modification in the cis-acting element. It is. Conversely, any modification, such as a deletion or insertion in a sequence region that can withstand such deletion or insertion without disrupting replication, is outside the cis-acting element. As shown herein, a substantial portion of an RNA viral molecule from which a substantial portion of an RNA viral molecule is prevented from replicating, using examples of BMVs known and accepted by those of skill in the art to be functional Without modification, they may be modified by deletion, insertion, or a combination of deletion and insertion.

“Exogenous RNA” is a term used to describe an RNA segment or component that is inserted into the viral RNA to be modified, where the source of the exogenous RNA segment is different from the RNA virus itself. The source of RNA may be an organism such as another virus, plant, animal, bacterium, virus or fungus. Exogenous RNA is chemically synthesized R
It may be NA, derived from natural RNA, or a combination of the above. Exogenous RNA may provide any function suitable and known to be provided by an RNA segment. Such functions include, but are not limited to, RNA that acts as a messenger RNA that, when translated by a host cell, results in the synthesis of a peptide or protein having a useful or desired trait. The RNA segment may be structural, eg, in ribosomal RNA; this may be regulatory, eg, with respect to small nuclear RNA, or antisense RNA; or catalytic It may be. One of skill in the art will appreciate that exogenous RNA, for example, may encode a protein that is a key enzyme in a biochemical pathway that, when expressed, exhibits a desired phenotypic characteristic, such as a change in cellular metabolism. It is. In addition, exogenous RNA may encode proteins involved in the regulation of transcription, such as zinc fingers, winged helix, and leucine zipper proteins. A particularly interesting function is provided by antisense RNA, sometimes referred to as (-) strand RNA, which indeed binds to and inhibits its function in target cells through complementary base pairing. A possible sequence is one that is actually complementary to another RNA sequence present in the target cell.

The term “non-viral” as used herein refers to any RNA segment not normally contained within the virus, whose modifications are utilized for replication and expression, and thus is referred to as “exogenous” Used synonymously with term. Thus, genes derived from a different viral species than the modified species are included within the meaning of the terms "non-viral" and "exogenous" for purposes of describing the present invention. For example,
Non-viral genes as the term include genes derived from bacterial, animal, or plant viruses of a type that can be distinguished from viruses that have been modified to effect transformation. Further, the non-viral gene may be a structural gene derived from any prokaryote or eukaryote.

In one embodiment, the invention relates to a novel method of transfecting host cells utilizing a DNA delivery platform to introduce viral RNA into cells. The present invention relates to a DNA encoding a modified viral RNA molecule, comprising an exogenous RNA component and an RNA viral component linked to a DNA-dependent RNA pol promoter.
Directed by a transfection method using a delivery platform. The DNA-dependent RNA pol promoter is preferably one of the modified viral RNA molecules.
It is fused within, but need not be, up to 10 nucleotides of the 'transcription start site, more preferably within 5 nucleotides of the 5' transcription start site. DNA
Expression of the delivery platform produces a transcript of the modified viral RNA molecule, which can then perform RNA replication in the presence of a replication factor, which replicates in the modified viral RNA. It may be provided in trans by other means, including present from and / or including expression from a chromosome, or may be provided on a different delivery plasmid. If the modified viral RNA is replicated, the exogenous RNA may be replicated as well. In addition, exogenous RNA can be expressed in cells, resulting in a defined phenotypic trait. In a preferred embodiment, the DNA delivery platform further comprises a nucleotide sequence encoding a self-cleaving ribozyme proximal to the 3 'end of the RNA molecule. As will be readily apparent to those skilled in the art, known ribozymes may be used in accordance with the present invention. In a preferred embodiment, the ribozyme cleaves a modified RNA viral molecule at the 3 'region. The 3 'region can be composed of up to 30 nucleotides upstream or downstream of the 3' end, and is preferably composed of up to 10 nucleotides upstream or downstream of the 3 'end. In a more preferred embodiment, the ribozyme cleaves the modified RNA viral molecule exactly at the 3 'end. Other known regulatory sequences, such as promoter and / or termination sequences, may also be substituted for and / or included on the DNA delivery platform. A suitable restriction enzyme site can be introduced proximal to the 3 'end of the modified viral RNA molecule sequence, and the DNA molecule can be cleaved with a suitable restriction enzyme prior to transfection. As used herein, "DNA-launching platform"
The term refers to a circular or linear DNA molecule that has a coding region that includes a segment that encodes a modified viral RNA segment, and that can further be transported into a cell and subsequently transcribed. It is interpreted as follows.

Possible regulatory sequences include promoters of viral origin (CaMV 19S and 35S) or so-called “housekeeping” genes (ubiquitin, actin, tubulin) with corresponding termination / polyA + sequences. It can include, but is not limited to, any promoter that has already been shown to be constitutive for expression. Similarly, plant fatty acid / lipid biosynthesis genes (ACP, acyltransferase, desaturase,
Targets seed and / or developmentally specific promoters, such as promoters of lipid transfer protein genes) or storage protein genes (eg, zein, napin, luciferin, conglycinin, phaseolin, or lectin genes) It can be used for chemical expression. In addition, the gene regulates an inducible promoter and its termination sequence such that gene expression is induced by light (rbcS-3A, cab-1), heat (hsp gene promoter), or wound (mannopine, HGPG). Can be put down. It will be apparent to those skilled in the art that the promoter may be used either in its native form or in a truncated form and may be paired with itself or a heterologous termination / polyA + sequence.

In a particularly preferred embodiment, the present invention is directed to a method of altering the genotype or phenotype of a cell, comprising the following steps: a) the viral RNA is the RNA product encoded thereby A viral RNA cDNA molecule, or RNA virus, that has been modified to include a DNA segment that encodes a non-viral RNA component located in a region that can withstand such insertions without interfering with the replication of the virus. Forming a cDNA molecule of at least one of the RNA components if is divided into many parts; b) cloning the modified cDNA into a DNA delivery platform; and c) placing the DNA in a suitable host cell. Transfecting the delivery platform. In a most preferred embodiment, the method further comprises the step of pre-transforming the plant with a trans-acting viral replication factor and / or other trans-acting factors. Such trans-acting factors may include viral motility proteins, coat proteins, viral proteases, and other structural and non-structural genes. In addition to stable expression of the trans-acting factor, the trans-acting factor may be introduced on a different expression plasmid or may be expressed from an RNA transcript. In a preferred embodiment, such trans-acting factors do not replicate. Suitable host cells may include protoplasts, cell suspensions, or cells in tissues or whole organisms.

[0028] In certain embodiments, which are to be construed as examples of the broader disclosure herein, the RNA viral segment can be derived from Poa annua moss mosaic virus (BMV), whereby the DNA delivery platform is a viral RNA3 Contains DNA encoding the segment. Spirulina moss mosaic virus (BMV) is a member of the alphavirus-like superfamily of animal and plant plus-strand RNA viruses and has a genome divided into three RNAs. RNA1 and RN2 encode 1a and 2a proteins, respectively, which are required for genomic RNA replication and subgenomic mRNA synthesis (eg, U.S. Patents, which are incorporated herein by reference to the extent not inconsistent with this specification). No. 5,500,360). These proteins are
It contains three domains that are conserved among all members of the virus-like superfamily. 1a (109 kDa) contains the c-proximal helicase-like domain and the n-proximal domain involved in RNA capping, and 2a (94 kDa) contains the central polymerase-like domain. For example, French and Ahlquist (1
988). 1a and 2a interact with each other and with cellular factors to form a membrane-associated viral RNA replication complex associated with the endoplasmic reticulum of infected cells. BMV RNA3, a 2.1 kb RNA, encodes a 3a protein (32 kDa) and a coat protein (20 kDa) that are involved in the transmission of BMV infection in natural plant hosts but are not important for RNA replication. See U.S. Patent No. 5,500,360. RNA
The 3a or coat protein gene of the three viral segments can be replaced by exogenous RNA, thereby not interfering with the replication element. In addition, an exogenous RNA segment can be inserted downstream of an additional subgenomic promoter. Still further, the cell or tissue contains a foreign gene with the required cis-acting components
The DNA delivery platform can be pre-transformed to express 1a, 2a, 3a, and coat proteins, or any combination thereof, that is transfected so that the foreign gene is replicated and / or expressed.

In one embodiment, the host cell is pre-transformed with BMV1 or BMV2 so that it is transgenically engineered to express the 1a and 2a proteins.
Preferably, the 5 ′ and 3 ′ ends of BMV1 and BMV2 are 1a and 2a because they cannot replicate, but form a viral RNA replication complex associated with the endoplasmic reticulum of the host cell.
Is excised to allow expression. Subsequent transfection of a DNA delivery platform containing the exogenous RNA of interest, along with the RNA3 viral replication segment, to express the exogenous RNA while preventing the unwanted and dangerous spread of viral RNA leaking into the environment Can be. That is, the plant
Since all three segments must be present in order to form infectious BMV particles, dangerous escape of foreign genes into the environment by mechanical or insect dissemination of the RNA viral vector is avoided. One of skill in the art will readily recognize that, in the BMV example, the DNA delivery platform may also be derived from either RNA1 or RNA2. For example, a sequence encoding the 1a protein can be replaced by exogenous RNA; replication requires expression of 1a (eg, a different expression plasmid). In a preferred embodiment, the DNA delivery platform also includes a ribozyme proximal to the 3 'end of the modified RNA3, wherein the ribozyme cleaves RNA3 at the 3' end. As will be readily apparent to one of skill in the art in light of the disclosure contained herein, viral segments and / or subviral material from other known viruses are used to formulate the DNA delivery platforms of the present invention. be able to. One skilled in the art will recognize that BMV is only a representative example of many viruses suitable for practicing the present invention. The principle on which the present invention is based
It is widely accepted that it is widely adaptable to countless viruses. Examples of other such viruses include alfalfa mosaic virus (AMV), wheat spot mosaic virus, cowpea mosaic virus, cucumber mosaic virus, reovirus, poliovirus, Sindbis virus, vesicular stomatitis virus, influenza virus, Including, but not limited to, retroviruses and cowpea chlorotic mottle virus (CCMV), and any other virus that can replicate through an RNA intermediate and obtain a cDNA copy therefrom. In particular, if one further characterizes other viruses, one of skill in the art will readily recognize that the disclosure herein is applicable to other suitable viruses.

[0030] One of skill in the art will readily recognize that known methods for introducing foreign genes into cells can be used in accordance with the disclosure of the present disclosure. Such methods include, but are not limited to, mechanical inoculation, particle-bombardment, agroinfection, electroporation, and microinjection, and other known methods.

[0031] Various aspects of the invention can be modified as needed, depending on the virus selected as the transformation and transfection agent and the specific properties of the RNA segment to be inserted. For example, the inserted gene need not be a naturally-occurring gene, may be modified, may be a complex of one or more coding segments, or may encode one or more proteins. Good. RNA can also be modified RNA
Modifications may be made by combining insertions and deletions to control the overall length or other properties of the molecule. The inserted non-viral gene may be of either prokaryotic or eukaryotic origin. The introduced gene may contain its own translation initiation signal, such as a ribosome binding site and an initiation (AUG) codon, or it may contain one or more of these components already present in the viral RNA to be modified You may insert so that it may utilize. Certain structural constraints must be observed in order to perform a correct translation of the inserted sequence according to principles well known in the art. For example, if an exogenous code segment is intended to be combined with an endogenous code segment, the coding sequence to be inserted must be inserted in its reading frame phase in the same translation direction.

The transfer does not cause a pronounced phenotypic alteration of the recipient cells,
It will be understood by those skilled in the art that certain genes may be present. Such may be the case, for example, when the translation product of a non-viral gene is toxic to the host cell and is degraded or treated to render it non-functional, or detectable on transformed cells. This can occur when the host cell has structural features that cannot be translated in sufficient amounts to confer a sufficient phenotype. However, the invention does not rely on any specific properties of the RNA segment or gene to be transferred. Thus, the possibility of the presence of RNA segments or genes that cannot confer the phenotypic trait readily observed on recipient cells or plants is
It is irrelevant to the present invention and in any case will be readily recognized by those skilled in the art without undue experimentation.

The exogenous RNA segment can be any convenient sequence of the viral RNA or cDNA sequence corresponding to the RNA component of a multipartite RNA virus, as long as the insertion does not interfere with sequences essential for replication of the RNA in the host cell. May be inserted into a well-inserted site. For example, in the case of a virus whose coat protein is not essential for replication, the exogenous RNA segment may be inserted within or normally replace the region encoding the coat protein. If desired, such a change may result in RN in the host cell.
Regions involved in undesired host cell responses may be deleted or inactivated as long as they do not adversely affect the replication ability of A. For many single-component and multi-component RNA viruses, a reduced normal rate of RNA replication is acceptable, and the amount of RNA produced during normal infection saturates the ribosomes of transformed cells It is preferable in some cases because it is more than sufficient to perform

Cultured plant cells are capable of replicating the RNA components expressed from the DNA delivery platform and thus dividing by cell division into progeny cells, so that as the cells proliferate and divide, It usually appears to be transfected. Plants regenerated from the phenotypically altered cells, tissues, or protoplasts are still phenotypically altered. Similarly, plants transfected as seedlings remain transfected during growth. The optimal timing for applying the transfected component will likely be governed by the results for which it is intended and by changes in sensitivity to the transfected component during various stages of plant development.

Many plant RNA viruses are transmitted by seed from one generation to the next. This property can be used to perform genotypic transformation of plants.
That is, the modified RNA remains transmissible from one generation to the next so that the seed-borne virus infection is transmitted from one generation to the next.

The following are examples illustrating techniques for practicing the present invention. These examples should not be construed as limiting. All percentages are weight percentages and all solvent mixing ratios are by volume unless otherwise specified.

Example 1 Construction of Agrobacterium Vector A binary vector expressing BMV 1a and 2a proteins in plants was constructed.
Start with the pB101.2 construct (Clontech, Palo Alto, CA). The GUS gene was excised by first cutting the construct with EcoRI and SnaBI. The ends of the overhang restriction fragments were plugged by treatment with Klenow fragment and dNTP. Re-ligate the end of the restriction fragment to pB101.2LR1
Was prepared.

[0038] The 2a expression cassette was inserted into pBI101.2LR1. First, pBI101.2LR1 was cut with HindIII and dephosphorylated. Next, pB2PA17 (Dinant et al., 1993)
Was cut with Hind III and the 2a insert was purified using a low melting point agarose gel. The ends of the restriction fragments were ligated to generate pB2LR4 and pB2LR5 (FIGS. 3c and 3d).

The 1a expression cassette was prepared by first cutting pBI101.2LR1 with SnaBI
It was inserted into I101.2LR1 and dephosphorylated. pB1PA17 (Dinant et al., 1993
) Was cut with PsI and extra nucleotides were removed by T4 DNA polymerase. The 1a insert was purified using a low melting point agarose gel. The ends of the restriction enzyme fragments were ligated to generate pB1LR2 and pB1LR3 vectors (FIGS. 3a and 3b).
).

The 1a expression cassette was inserted into pB2LR2 and pB2LT5 by cutting pB2LR4 or pB2LR5 with SnaBI and dephosphorylated. pB1PA17 (Dinanto
t) et al., 1993) were cut with PstI and extra nucleotides were removed with T4 DNA polymerase. The 1a insert was purified using a low melting point agarose gel,
Ligation with the cut pB2LR4 or pB2LR5 vector, pB12LR6, pB12L
The R7, pB12LR8, and pB12LR9 vectors were generated (FIGS. 3e-3h).

Example 2-DNA delivery platform for wtRNA3 of BMV, including exogenous sequences
Construction of DNA Delivery Platform for RNA Derivatives The vector pRT101 (Topfer et al., 1987) was cut with PpuMI, the ends of the restriction fragments were cleaved with Klenow fragment and dNTP, cut with BamHI and dephosphorylated. The vector pB3RQ39 (Ishikawa et al., 1997) was transformed with SnaBI and BamH.
Cleavage by I; B3 fragment was isolated from a low melting point agarose gel. Disconnected p
This fragment was ligated into RT101, thereby creating pB3LR10 (FIG. 4). The pB3LR10 derivative pB3LR15 (FIG. 4) has a ClaI-KpnI fragment (Janda et al., 1987) replaced by the corresponding fragment from pB3TP8.

PCR was performed on pRT101 to amplify EcoRV and EcoRI fragments. A single nucleotide deletion was made during the PCR process to create a StuI site instead of a PpuMI site. The resulting PCR product was cut with EcoRV and EcoRI and inserted into pRT101 that had been cut with EcoRV and EcoRI and dephosphorylated to create pRT101LR11. pRT101LR11 was cut with StuI and BamHI and dephosphorylated. PB3RQ
39 was cut with SnaBI and BamHI, and the B3 fragment was isolated using a low melting point agarose gel. Next, the fragment was ligated to pRT101LR11 to produce pB3LR12 (FIG. 4).

Another DNA delivery platform was constructed by wtRNA3 of BMV with a partially double-stranded CaMV35S promoter; thereby pB3LR14 and pB3LR16
(FIG. 4).

A DNA delivery platform in which the BMV RNA3 coat protein was replaced by GUS was similarly constructed. pB3MI22 (Ishikawa et al., 1997) was cut with ClaI and StuI to isolate the B3GUS insert. The pB3LR10 or pB3LR14 DNA delivery platform construct was cut with ClaI and StuI and dephosphorylated. Next, the B3GUS fragment was ligated to the cleaved pB3LR10 or pB3LR14, thereby creating the pB3GUSLR17 and pB3GUSLR18 DNA delivery platform constructs (FIG. 5).

A DNA delivery platform was constructed with GUS gene inserted BMV RNA3, with GUS downstream of an additional BMV subgenomic promoter. AvaI construct for pB3LR15
And the end of the restriction fragment was blocked with Klenow fragment and dNTP. Next, the construct was cut with ClaI and dephosphorylated. pB3MI22 was cut with ClaI and StuI to isolate the B3GUS fragment. Next, the isolated B3GUS fragment was ligated to the cleaved pB3LR15 construct to create a new construct of pB3GUSCPLR19 (FIG. 5).

A BMV RNA3-based DNA delivery platform was constructed with the CP gene inserted downstream of an additional cowpea chlorotic mottle virus (CCMV) subgenomic promoter. The pB3GUSLR17 construct was cut with StuI and KpnI and dephosphorylated. pBC3AJ14 (Pacha and Ahlquist, 1991)
Was cut with NdeI, the ends were made blunt by methods known in the art, and cut with KpnI. Next, the coat protein fragment was isolated. The coat protein fragment was ligated to the cut pB3GUSLR17 to create a new construct of pB3GUSCPLR22 (FIG. 5).

A DNA delivery platform with subgenomic RNA4 was constructed. pB4MK2 (M. Kroll, personal communication) was cut with SnaBI and BamHI to isolate the RNA4 fragment. The pRT101LR11 construct was cut with StuI and BamHI and dephosphorylated. The fragment and the truncated pRT101LR11 construct were ligated to generate pB4LR20 (FIG. 5a).

A DNA delivery platform in which the BMV coat protein was replaced with GFP was constructed. pEG
FP (Clontech, CA) was cut with NotI, plugged with Klenow fragment and dNTP, and the GFP insert was isolated using a low melting point agarose gel. pB3LR15 was cut with SalI and StuI and dephosphorylated. Next, GFP
The fragment was ligated into the cut pB3LR15, thereby creating pB3GFPLR48 (FIG. 6e).

A DNA delivery platform with BMV RNA3 inserted with the GFP gene, where the CP was downstream of an additional CCMV subgenomic promoter, was constructed. pBC3AJ14 (Pacha &
Earlquist (Pacha and Ahlquist, 1991) was cut with NdeI and EcoRI and the ends were blunted by methods known in the art. Next, the coat protein fragment was isolated, dephosphorylated, blunted, and ligated to NoE and StuI cut pEGFP to create pEGFPCPLR49. pEGFPCPLR49 was cut with KpnI and the EGFPCP fragment was isolated using a low melting point agarose gel. PB3GFPLR48 to KpnI
And dephosphorylated. Next, the EGFPCP fragment was ligated into the cleaved pB3GFPLR48, thereby creating pB3GFPCPLR50 (FIG. 6a).

An RNA transcription vector in which the GFP gene was expressed as a translational fusion with BMV 3a was constructed. pB3TP10 (Pacha and Ahlquist, 1991) with BamHI
And dephosphorylated. PEGFP (using PCR and the following primers)
GFP fragment from Clontech, CA). The amplification product was cut with BamHI and purified using a low melting point agarose gel.
The GFP fragment was ligated to the cleaved pB3TP10 to produce pB3GFPLR47 (see FIG.
6d). pB3GFPLR47 was cut with EcoRI and transcribed using T7 RNA polymerase.

An Agrobacterium vector containing the BMV RNA3 DNA delivery platform was constructed. pBI101.2LR1 was cut with SmaI and dephosphorylated. pB3LR15 was cut with PvuII and the B3 fragment was purified using a low melting point agarose gel. Next, the B3 fragment was ligated into pBI101.2LR1, thereby creating pB3LR42 (FIG. 9).
.

[0052] BMV RNA3 coat protein was converted to SHMV (Sunn hemp mosaic virus).
A DNA delivery platform was constructed that replaced the coat protein and inserted the GUS gene downstream of an additional BMV subgenomic promoter. pB3RS4 (Sac
her) et al., 1988) were cut with AvaI, blunt-ended with Klenow fragment and dNTP, and cut with KpnI. SHMV coat protein fragments were isolated using low melting point agarose gels. pB3GUSLR17 was cut with StuI and KpnI and dephosphorylated. The SHMV coat protein fragment was ligated to the cut pB3GUSLR17, thereby producing pBGUSCPLR24 (FIG. 7).

A DN comprising one or more foreign genes and a required cis-acting replication signal
Other permutations of the A delivery platform will be readily apparent upon reviewing the disclosure herein. See, for example, Examples 5-10.

Example 3 Tobacco (N. tabacum) Protopras with DNA Delivery Platform
G1 transfection medium: NT1 medium (1 liter) contains Gibco-BRL (MS salt, catalog number 11118-031), 6
Prepared with 3 ml of% KH ₂ PO ₄ and 0.2 μg / ml of 2,4D (final concentration). pH is KOH
The mixture was adjusted to 5.5 to 5.7 using, and the resulting mixture was autoclaved.

An NT1 seeding medium (1 liter) was prepared from the NT1 medium and 72.86 g of mannitol, the pH was adjusted to 5.5 to 5.7, and the resulting mixture was autoclaved.

A washing solution (1 liter) was prepared with 72.86 g of mannitol, the pH was adjusted to 5.5, and the resulting mixture was autoclaved.

The electroporation buffer was 0.8% NaCl, 0.02% KCl, 0.02% KH ₂ PO ₄ , 0.
Made from 11% Na ₂ HPO ₄ and 0.4 M mannitol. The pH was adjusted to 6.5 and the resulting mixture was autoclaved.

An enzyme solution was made from 0.4 M mannitol and 20 mM MES. The pH was adjusted to 5.5 and the resulting mixture was autoclaved.

Growth conditions: Cells (tobacco (Nicotiana tabacum)) were grown in NT1 medium at room temperature under constant shaking conditions (about 200 rpm).

Preparation of Culture for Digestion: About 2-3 ml of the 1-week suspension culture was added to fresh NT1
After subculture into 50 ml of the medium and culturing for 3 days, enzyme digestion was performed. Cultures were maintained at 28 ° C. under constant shaking conditions.

Enzyme digestion: An enzyme digestion solution containing the following was prepared: an enzyme solution of 1% serridine (Calbiochem) and 0.3% macerase (Calbiochem). pH 5
Adjusted to .5 and sterilized by filtration.

The cells were centrifuged at 800 rpm for 5 minutes. The supernatant was discarded. The cells were resuspended by adding about 40 ml of wash solution and centrifuged at 800 rpm for 5 minutes. Next, three volumes of cells were resuspended in the enzyme digest and incubated at room temperature for 60 minutes.

Washing: Cells were transferred to a 50 ml plastic tube and centrifuged at 800 rpm for 5 minutes. The supernatant was discarded. Cells were resuspended in 40 ml of wash and centrifuged at 800 rpm for 5 minutes. The supernatant was discarded. Cells were resuspended in 40 ml of electroporation buffer and centrifuged at 800 rpm for 5 minutes. The supernatant was discarded. Cells were resuspended in 4 volumes of electroporation buffer.

Electroporation: 1 ml of cells containing RNA or DNA inoculum was transferred to an electroporation cuvette and left on ice for 10 minutes. The cells were then mixed and electroporated at 500 μF, 250 V. The cuvette was placed on ice for 10 minutes. Cells were transferred to 10 ml of NT1 seeding medium.

Sample Incubation and Collection: Cells were incubated in the dark at room temperature. Samples were collected 24-48 hours after inoculation.

RNA analysis: RNA extraction, denaturing 1% agarose gel electrophoresis, and Northern blot hybridization were performed by Rasochova and Miller (1996).
) Was performed by known methods. Each lane was loaded with an equal amount (approximately 5 μg) of total RNA measured by spectrophotometry,
RNA was confirmed by ethidium bromide staining before Northern blot hybridization. 1 × 10 ⁶ cp per hybridization experiment
A hybridization buffer solution containing m / ml radioactive probe was used. RNA3
Replication was confirmed by detection of sgRNA4, thus, BMV RNA replication factors 1a and 2a expressed from the expression plasmid were supplied as in vitro transcripts (FIG. 11).
) Together with the DNA delivery platform (FIG. 12), indicating that it supports efficient replication of RNA3.

Example 4 Preparation of Transgenic Tobacco (N. tabacum) Plant After the desired molecule was constructed in E. coli, the molecule was transferred to Agrobacterium tumefaciens using a freeze-thaw method.
The vectors pB1LR2, pB2LR4, pB12LR6, and pB12LR7 were all used individually. Agrobacterium LBA 4404 containing the appropriate helper Ti plasmid was added to 5 ml of YEP medium.
Grow overnight at 8 ° C. 2 ml of the overnight culture was added to 50 ml of YEP medium in a 250 ml flask and the culture was shaken vigorously at 28 ° C. until the OD ₅₀₀ grew from 0.5 to 1.0 (250
rpm). The culture was cooled in ice. The cell suspension was centrifuged at 3000 g for 5 minutes at 4 ° C. The supernatant was discarded. Cells were resuspended in 1 ml of ice cold 20 mM CaCl ₂ solution. 0.1 ml aliquots were transferred to pre-cooled Eppendorf tubes. About 1μg of plasmid DNA
Was added to the cells. Cells were frozen in liquid nitrogen. Cells were lysed by incubating the tubes in a 37 ° C. water bath for 5 minutes. 1 ml of YEP medium was added to the tube and incubated at 28 ° C for 2-4 hours with gentle shaking to allow the bacteria to express the antibiotic resistance gene. The tubes were centrifuged for 30 seconds and the supernatant was discarded. Cells were resuspended in 0.1 ml of YEP medium, seeded on YEP agar plates containing the selected antibiotic and incubated at 28 ° C. Transformed colonies appeared in 2-3 days.

An in vitro clone copy of about 3 weeks old tobacco (Nicotiana tabacum), Wisconsin # 38 was used as the explant source. Leaf explants were prepared from second and third fully grown leaves in in vitro culture. Leaf pieces were cut into 1 cm × 1 cm squares and placed on TB1 (supplemented with 2.0 mg / L 6-benzyl-aminopurine and 0.1 mg / L 1-naphthaleneacetic acid) medium at 25 ° C. for 16 hours under lighting conditions. Left for hours.

Agrobacterium tumefaciens containing a preselected binary vector
LBA 4404 strain was used for plant transformation. Explants were placed in ~ 10 ml of Agrobacterium culture grown overnight for 30 minutes. Absorb the moisture from the leaf explants with filter paper and add
(1.0 mg / L 6-benzyl-aminopurine and 0.1 mg / L 1-naphthaleneacetic acid were added.) The medium was left standing for 4 days with its abscissa face down. Next, the explants were rinsed three times with sterile water, blotted on filter paper and placed on a TB2 medium containing 100 mg / L kanamycin and 400 mg / L carbenicillin, abaxial down, at 25 ° C. For 16 hours under light conditions. Explants were transferred to fresh TB2 medium containing 100 mg / L kanamycin and 400 mg / L carbenicillin every 10-14 days until they grew into plants. Plants typically grew in 10-14 days. Plants were cut from calli and added to MST medium containing 100 mg / L kanamycin and 400 mg / L carbenicillin to induce rooting. Rooted plants were transferred to soil.

In TB1 (1 liter), MS salt 4.30 g, myo-inositol 100 mg, Nitsch and Nitsch vitamin 1.0 ml, sucrose 30 g, BAP 2 mg, NAA 0.10
mg, and 8 g of Noble Agar. The medium was adjusted to pH 5.7 and autoclaved.

In TB2 (1 liter), MS salt 4.30 g, myo-inositol 100 mg, NITCH &
It contained 1.0 ml of niche vitamin, 30 g of sucrose, 1.0 mg of BAP, 0.10 mg of NAA, and 8 g of Noble Agar. The medium was adjusted to pH 5.7 and autoclaved.

MST (1 liter) contains MS salt 4.30 g, Nitsch & Nitsch vitamin 1.0 ml,
It contained 30 g of sucrose, 100 mg of myo-inositol and 8.5 g of Difco agar. The medium was adjusted to pH 5.7 and autoclaved.

YEP (100 ml) contains 1.0 g of bacto peptone, 1.0 g of bacto yeast extract, and N
0.5 g of aCl was included. The medium was autoclaved.

RNA analysis: Total RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization were performed by Rasochova and Miller (1996).
) Was performed by known methods. Each lane was loaded with an equal amount (approximately 5 μg) of total RNA measured by spectrophotometry,
RNA was confirmed by ethidium bromide staining before Northern blot hybridization. 1 × 10 ⁶ per hybridization experiment
A hybridization buffer solution containing a cpm / ml radioactive probe was used. FIG.
a shows successful expression of BMV 1a and 2a mRNA in transgenic tobacco (N. tabacum).

Example 5 Transgenic Tobacco (N. taba with DNA Delivery Platform)
cum) Transfection particles of plants- microcarriers for bombardment
Deposition of DNA on: (Kikkert, 1993).

Microcarrier sterilization: 80 mg of gold particles were resuspended in 1 ml of 70% ethanol, soaked for 15 minutes, and centrifuged at 13,000 × g for 5 minutes. The supernatant was carefully removed and discarded. The particles were resuspended in 1 ml of sterile distilled deionized water and centrifuged at 13,000 xg for 5 minutes. The supernatant was carefully removed and discarded. Further, the washing of the particles with water was repeated twice. After the last rinse, the particles were resuspended in 1 ml of sterile 50% glycerol.

Coating of microcarriers with DNA: The following were added continuously and quickly: 5 μl of DNA (1 μg / μl), 50 μl of 2.5 M CaCl ₂ , and 0.1 M spermidine 20
μl.

The mixture was incubated on a vortex shaker at room temperature for 10 minutes. The particles were spun down at 13,000 × g for 5 seconds. The supernatant was carefully removed and discarded. The particles were resuspended in 140 μl of 70% ethanol and centrifuged at 13,000 × g for 5 seconds. The supernatant was removed and discarded. The particles were resuspended in 140 μl of 100% ethanol and centrifuged at 13,000 × g for 5 seconds. The supernatant was removed and discarded. The particles were resuspended in 50 μl of 100% ethanol.

[0079] PDS1000He biolistic gun (DuPont) and 1100 psi rupture disc (Bio-Rad) were applied to young leaves of tobacco plants grown in vitro on MS agar solid media containing 30 g / L sucrose. ) Was used to bombard a 5 μl aliquot of the resuspended DNA-coated particles.

RNA analysis: Total RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization were performed by Rasochova and Miller (1996).
) Was performed by known methods. Each lane was loaded with an equal amount (approximately 5 μg) of total RNA measured by spectrophotometry,
RNA was confirmed by ethidium bromide staining before Northern blot hybridization. 1 × 10 ⁶ per hybridization experiment
A hybridization buffer solution containing a cpm / ml radioactive probe was used. Fig. 14
a is that the delivered BMV RNA3 replicates efficiently in transgenic plants expressing BMV replication factors 1a and 2a, and that the delivered RNA3 is BMV 1a and / or
This indicates that replication is not possible in the absence of 2a.

Example 6-Transgenic Nicotiana benthami
ana) Preparation of plant After the desired molecule was constructed in E. coli, the molecule was introduced into Agrobacterium tumefaciens. The vectors pB1LR2, pB2LR4, pB12LR6, and pB12LR7 were all used individually. Agrobacterium LBA 4404 containing the appropriate helper Ti plasmid
Grow at 28 ° C. in 5 ml of YEP medium. Transfer 2 ml of the overnight culture into a 250 ml flask with YE
In addition to 50 ml of P medium, the culture was shaken vigorously at 250C until the OD ₅₀₀ grew from 0.5 to 1.0 (250 rpm). The culture was cooled in ice. Cell suspension at 3000C for 5 g
Centrifuge for minutes. The supernatant was discarded. Cells were resuspended in 1 ml of ice cold 20 mM CaCl ₂ solution. 0.1 ml aliquots were transferred to pre-cooled Eppendorf tubes. About 1 μg of plasmid DNA was added to the cells. Cells were frozen in liquid nitrogen. Cells were lysed by incubating the tubes in a 37 ° C. water bath for 5 minutes. YEP medium 1
The ml was added to the tube and incubated at 28 ° C. for 2-4 hours with gentle shaking to allow the bacteria to express the antibiotic resistance gene. The tube was centrifuged for 30 seconds and the supernatant was discarded. Cells were resuspended in 0.1 ml YEP medium. Y with selected antibiotics
Cells were seeded on EP agar plates and incubated at 28 ° C. Transformed colonies appeared in 2-3 days.

An in vitro clonal copy of N. benthamiana, about 5-7 weeks of age, was used as an explant source. Leaf explants were prepared from second and third fully grown leaves in in vitro culture. Cut leaf pieces into 1cm x 1cm squares,
The plate was left on a MS104 medium at 23 ° C. for 16 hours under illumination conditions for 24 hours in a 0 × 15 mm plate.

Agrobacterium tumefaciens containing a preselected binary vector
LBA 4404 strain was used for plant transformation. Explants were placed in ~ 10 ml of Agrobacterium culture grown overnight for 30 minutes. Wipe off the moisture from the leaf explants with filter paper, and MS1
The medium was allowed to stand in the medium 04 with its abaxial surface facing down for 4 days. The explants were then rinsed three times with sterile water, blotted on filter paper and regenerated on MS104 medium containing 300 mg / L kanamycin and 400 mg / L carbenicillin. Explants grow 10-1 until they grow into plants
Fresh MS104 containing 300 mg / L kanamycin and 400 mg / L carbenicillin every 4 days
Transferred to medium. Plants typically grew between 31 and 50 days. Plants were cut from calli and placed on MST medium containing 300 mg / L kanamycin and 400 mg / L carbenicillin to induce rooting. Rooted plants were transferred to soil.

In 1 liter of MS104, 4.3 g of MS salt mixture, 1.0 ml of B5 vitamin solution, 30 g of sucrose
, BA 1.0 mg, NAA 0.1 mg, and phytagger 8.0 g. The medium was adjusted to pH 5.8 and autoclaved.

In 100 ml of YEP medium, 1.0 g of bacto peptone and 1.0 g of bacto yeast extract, NaC
l 0.5 g was included. The medium was autoclaved.

In 1 liter of MST, 4.3 g of MS salt mixture, 1.0 ml of Nitsch & Nitsch vitamin
, Sucrose 30 g, myo-inositol 100 mg, and phytager 8.5 g. The medium was adjusted to pH 5.7 and autoclaved.

RNA analysis: Total RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization were performed by Rasochova and Miller (1996).
) Was performed by known methods. Each lane was loaded with an equal amount (approximately 5 μg) of total RNA measured by spectrophotometry,
RNA was confirmed by ethidium bromide staining before Northern blot hybridization. 1 × 10 ⁶ per hybridization experiment
A hybridization buffer solution containing a cpm / ml radioactive probe was used. FIG.
b shows successful expression of BMV 1a and 2a mRNA in transgenic N. benthamiana.

Example 7-Transgenic N. benthamiana plant tiger
Deposition of DNA on Microcarriers for Transfection Particle-Bombardment: (
Kikkert, (1993) “Biolistic PDS 1000 / He instrument (The
biolistic PDS 1000 / He device) ", Plant Cell Tiss. And Org. Cult. 33: 2
21-226).

Sterilization of microcarriers: 80 mg of gold particles were resuspended in 1 ml of 70% ethanol, soaked for 15 minutes, and centrifuged at 13,000 × g for 5 minutes. The supernatant was carefully removed and discarded. The particles were resuspended in 1 ml of sterile distilled deionized water and centrifuged at 13,000 xg for 5 minutes. The supernatant was carefully removed and discarded. Further, the washing of the particles with water was repeated twice. After the last rinse, the particles were resuspended in 1 ml of sterile 50% glycerol.

Coating of microcarriers with DNA: The following were added continuously and rapidly: 5 μl of DNA (1 μg / μl), 50 μl of 2.5 M CaCl ₂ , and 0.1 M spermidine 20
μl.

The mixture was incubated on a vortex shaker at room temperature for 10 minutes. The particles were spun down at 13,000 × g for 5 seconds. The supernatant was carefully removed and discarded. The particles were resuspended in 140 μl of 70% ethanol and centrifuged at 13,000 × g for 5 seconds. The supernatant was removed and discarded. Resuspend the particles in 140 μl of 100% ethanol and add 13,000 xg
For 5 seconds. The supernatant was removed and discarded. The particles were resuspended in 50 μl of 100% ethanol.

[0092] Young leaves of N. bentamiana plants grown in vitro on MS agar solid media containing 30 g / L sucrose were treated with a PDS1000He biolistic gun (DuPont) and a 1100 psi rupture disk (DuPont). Biorad) was used to bombard a 5 μl aliquot of the resuspended DNA coated particles.

RNA analysis: Total RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization were performed by Rasochova and Miller (1996).
) Was performed by known methods. Each lane was loaded with an equal amount (approximately 5 μg) of total RNA measured by spectrophotometry,
RNA was confirmed by ethidium bromide staining before Northern blot hybridization. 1 × 10 ⁶ per hybridization experiment
A hybridization buffer solution containing a cpm / ml radioactive probe was used. The delivered BMV RNA3 replicates efficiently in transgenic N. bentamiana plants expressing BMV replication factors 1a and 2a (FIG. 14b), and in the absence of BMV 1a and / or 2a Indicated that it cannot be duplicated.

Example 8 Transgenic Plants by GUS Containing a DNA Delivery Platform
Transfection transgenic tobacco (N. tabacum) and N. bentamiana (N. bentam)
iana) Plants were produced according to the method described above. DNA containing the GUS gene (FIG. 5a) was added to plants by particle-bombardment as described in Examples 5 and 7.
The delivery platform was transfected. The plants were incubated for 3-5 days and 1 M / ml X-Gluc (5-bromo-4-chloro-3-indolyl glucuronide) as a substrate, 0.1 M potassium phosphate buffer, pH 7.0, 50 μM potassium ferrocyanide, And assayed for β-glucuronidase (GUS) activity in 2% Triton® X-100. After overnight incubation at 37 ° C, cells expressing the delivered RNA3 derivative and expressing the GUS reporter gene from subgenomic RNA4 produced blue spots (Figure 15). The delivered RNA3 derivative is BMV RNA replication factor 1a
And did not express the GUS reporter gene (eg, wild-type N. bentamiana and wild-type tobacco (N. tabac).
um)).

Example 9 Transgenic Plant Tigers Expressing BMV 1a, 2a, 3a, and CP
The transfected plants were transformed with the BMV 1a, 2a, 3a and CP genes, thereby stably expressing those genes in the plants. This can be done by the method outlined above. Any necessary modifications will be readily apparent to one skilled in the art in light of the disclosure contained herein. The RNA comprising a BMV or CCMV cis-acting replication signal required to replicate foreign genes and RNA replicons
A DNA delivery platform encoding the replicon is constructed (FIG. 10b). The foreign gene contained in the replicon can include, for example, a Bacillus thuringiensis polynucleotide encoding a Bt protein.
Other sequences, such as those encoding herbicide resistance, or any known sequence encoding a peptide or protein having the desired properties in the plant are also included.

Alternatively, plants can be transformed to express BMV 1a, 2a, 3a and TMV coat protein instead of BMV coat protein. Next, one or more foreign genes, the required cis-acting replication signals of either BMV or CCMV, and T
Create a DNA delivery platform containing the MV origin of assembly (FIGS. 8a, 8b, and 10a). This delivery platform has distinct advantages because TMV is a rod-shaped virus that does not strictly limit the size of the RNA that can be encapsulated. Alternatively, TMV motor protein can be used instead of BMV3a (FIG. 7c). Hybrids with tobamo and bromovirus have been shown to survive (Sacher et al., 1988; Dejon & Earlquist (De
Jong and Ahlquist), 1992).

[0097] Other permutations and combinations of pre-transformed genes and genes included in the DNA delivery platform will be readily recognized by those of skill in the art in light of the disclosure herein (eg, FIG. 8c). , 10b, and 10c).

[0098] As described above, the CCMV subgenomic promoter can be replaced with a BMV sequence in the desired DNA delivery platform. Since the sequence of the CCMV subgenomic promoter is different from the sequence of the BMV subgenomic promoter, the recombination potential for loss of the foreign gene is much lower for constructs having a combination of these two different promoters.

In the above examples, the trans-acting component is a component such as a replication factor, a component involved in intercellular motility, or a coat protein that may be required for long distance propagation, It may include, but is not limited to, involved viral proteases, or other known trans-acting functions.

Example 10-Tobacco (N. tabacum) Produced by GUS-Containing DNA Delivery Platform
Transfection of Toplasts Tobacco (N. tabacum) protoplasts isolated using the method described above were inoculated by electroporation with a DNA delivery platform for BMV RNA3 derivatives in the presence and absence of 1a and 2a expression plasmids. . BMV RNA3 derivatives contain the GUS gene instead of the coat protein ORF (FIG. 5a) (these were inoculated with or without the coat protein expression plasmid (FIG. 5b)) or
Or having a BMVCP gene translated from an additional subgenomic RNA derived from the CCMV subgenomic promoter (FIGS. 5c and 5d), or an additional BMV subgenomic RN
It had SHMV coat protein translated from A (FIG. 7b). Protoplast inoculation 2
Four hours later, the cells were collected by centrifugation (800 rpm, 5 minutes). The chemiluminescent GUS assay is
Performed using GUS-Lite® (Tropix, Mass.) According to the manufacturer's instructions. Protein concentration was measured using a Bio-Rad protein kit (Bio-Rad Laboratories, Hercules, CA). GUS values measured by the luminometer were adjusted to the same total protein concentration. FIGS. 16a and 16b show successful GUS expression in protoplasts in the presence of trans-acting BMV replication factors 1a and 2a.

Example 11-Tobacco (N. tabacum) plants with GFP containing DNA delivery platform
Transfection of rotoplasts Tobacco (N. tabacum) protoplasts isolated using the above method were added to an expression plasmid for trans-acting BMV replication factors 1a and 2a, and a BMV coat protein OR
DNA delivery platform for RNA3 derivatives with the GFP gene in place of F (FIG. 6e), CP gene translated from additional subgenomic RNA (FIG. 6a), or BMV 3
a RNA transcript with GFP expressed as a fusion protein with the ORF (FIG. 6d) was transfected by electroporation. Protoplasts were incubated for 24 hours and examined for GFP expression using a fluorescence microscope. FIG. 18 shows successful expression of GFP in protoplasts.

Example 12-DNA delivery platform based on BMV RNA3 containing GFP (1a +
2a) Transfection of transgenic plants BMV RNA3 having a GFP gene instead of a BMV coat protein was added to N. bethamiana plants using particle-bombardment as described above.
The DNA delivery platform was transfected (FIG. 6e). GFP expression was measured 24 hours after inoculation using a fluorescence microscope. FIG. 17 shows successful expression of GFP in (1a + 2a) transgenic N. bethamiana.

Example 13-BMV RNA3 DNA delivery platform using Agrobacterium
(1a + 2a) Transgenic N. bethamiana plant
The transfection N. benthamiana (N. bethamiana) plants were inoculated with BMV RNA3 DNA delivery platform using Agrobacterium tumefaciens. The desired construct (
After pB3LR42) was obtained in E. coli, it was introduced into the A. tumefaciens strain using the freeze-thaw method as described above. Agrobacterium was grown overnight at 28 ° C under constant shaking conditions. N. bethamiana
One of the leaves at the bottom of () was pierced several times with a needle and immersed in an Agrobacterium culture solution.
Plants were grown at 23 ° C. for 16 hours under light conditions. The inoculated leaves were collected 14 days after inoculation. Total RNA extraction and Northern blot hybridization were performed as described above. FIG. 19 shows inoculated (1a + 2a) transgenic N. benthamiana (N
bethamiana) is shown.

Example 14-DNA delivery platform based on BMV RNA3 containing GUS and SHMV coat proteins
Transfection of (1a + 2a) Transgenic Plants by Foam In a N. bethamiana plant, the BMV coat protein is replaced with SHMV coat protein (San hemp mosaic virus) using particle-bombardment as described above. A transfected DNA delivery platform for BMV RNA3, in which the GUS gene was inserted downstream of an additional BMV subgenomic promoter (FIG. 7b). GUS expression was determined by histochemical GUS assay as described above. FIG. 20 shows successful GUS expression in (1a + 2a) transgenic plants.

Example 15-Exercise of delivered BMV RNA3 Self-pollinated (1a + 2a) transgenic N. benthamian
a) BMV R was added to F1 progeny plants of BP14 using Agrobacterium tumefaciens.
The NA3 DNA delivery platform was inoculated. Seedlings were germinated on Smurf medium containing kanamycin. Plants were grown at 23 ° C. for 16 hours under light conditions. After the desired construct (pB3LR42) has been obtained in E. coli, it can be purified using the freeze-thaw method as described above.
Introduced into Tumefaciens LBA 4404 strain. Agrobacterium was grown overnight at 28 ° C under constant shaking conditions. One leaf of the lower leaf of N. bethamiana was pierced several times with a needle and immersed in an Agrobacterium culture solution. The inoculated middle and upper leaves were collected 14 days after inoculation. Total RNA extraction and Northern blot hybridization were performed as described above. RNA3 replication was detected in all leaves examined (FIG. 21). This is in (1a + 2a) transgenic plants
It indicates that BMV RNA3 is capable of replicating, capable of intercellular movement, and capable of propagating long distances.

Example 16- (1a + 2a) Transgenic N. bethamiana
Transfection of their offspring with the BMV RNA3 DNA delivery platform Self-pollinated with Agrobacterium as described in Example 13 (1a +
2a) Transgenic N. benthamiana (designated as BP14) contains BMV RNA3 DNA
The delivery platform was inoculated. The control plants (non-transgenic N. benthamiana) were inoculated juice BMV infected barley with inoculation buffer consisting of 50 mM NaPO _4, pH 7.0 and 1% celite. Root samples were collected 6 weeks after inoculation. R
NA extraction and Northern blot analysis were performed as described above. Figure 22 shows BM at the root
V RNA3 replicated to very high levels. Some (1a + 2a
) In transgenic plants (Figure 22, lanes 2,5,6,7,8,10), the replication of the delivered RNA3 was reduced by wild-type B in non-transgenic N. benthamiana plants.
It greatly exceeded the replication of MV (FIG. 22, lane 1). This means that this system can be used to transport RNA, proteins, peptides or other compounds to the roots,
And that such compounds can be tested for various activities, such as those directed against root parasites. For example, a protein having anti-nematode activity can be inserted into the RNA3 DNA delivery platform using the strategy described above, and RN
When A3 replicates, it can be expressed in roots. Such proteins can be engineered to be expressed in the cytoplasm or secreted into the surrounding soil.

Example 17-Barley spotted leaf mosaic virus Barley spotted leaf mosaic virus (BSMV) has a three-part genome (
RNAα, β, and γ). These genomic RNAs have an m7Gppp cap and a 3 '
Has a t-RNA-like structure at the end (Jackson and Hunter, 1
989).

DNA delivery plasmids for BSMV RNAα, RNAβ, and RNAγ, including BSMV RNA cDNA, were constructed by precise fusion to a DNA-dependent RNA polymerase promoter at the 5 ′ end and a self-cleaving ribozyme at the 3 ′ end. You. A polyadenylation signal may also be included. Alternatively, a convenient restriction enzyme site may be
It may be manipulated at the 3 'end. A foreign gene or sequence may be expressed in several ways. For example, a DNA delivery plasmid based on BSMV RNAβ is a foreign gene, or ORFβ
It may include a sequence to be expressed instead of a.

A transgenic plant is obtained in which one or more trans-acting factors are fused with a DNA-dependent RNA polymerase promoter and terminator. Such trans-acting factors may include part of the viral RNA replicase (ORFαa and / or γa), or other trans-acting factors. The trans-acting factor is stably expressed in plant cells, or when using an inducible promoter,
Its expression may be induced. The cis-acting sequences required for BSMV RNA replication are excised from the transgene. Alternatively, full-length RNAα is expressed from the chromosome. Alternatively, ORFγ, including a 5 ′ untranslated region such as ND18 and ORFγb, from a seed propagation strain is also expressed (Edwards, 1995).

The DNA-dependent RNA polymerase promoter fused exactly to the 5 ′ end of BSMV RNAβ, cis-acting elements such as the 5 ′ and 3 ′ ends important for the life cycle of BSMV RNAβ, between the βa and βb ORFs A DNA delivery plasmid is constructed that contains the intercistronic region (Zhou and Jackson, 1996) and a foreign gene or sequence in place of ORFβa (coat protein) that is not important for BSMV replication and movement. Such DNA delivery plasmids may lack the internal poly (A) region because this region is not important by replication,
A ribozyme or convenient restriction enzyme site may be included at the 3 'end of the NA. Alternatively, the DNA delivery plasmid has ORFγa and / or γb, three gene block genes (ORFβb, βc, and βd), or a heterologous motor protein (TMV 30K, RCNMV35K)
Is constructed from RNAγ which has been replaced by a foreign gene or sequence.

Example 18-Tobacco mosaic virus Tobacco mosaic virus (TMV) has a single-stranded plus-sense RNA genome. The 5 'end has an m7Gppp cap and the 3' end contains a t-RNA-like structure.

A TMV RNA-based DNA delivery plasmid is constructed that contains the TMV cDNA fused exactly at the 5 ′ end to the DNA-dependent RNA polymerase promoter and fused at the 3 ′ end to a self-cleaving ribozyme. A polyadenylation signal may be included as well. Alternatively, a convenient restriction enzyme site may be engineered at the 3 'end. The foreign gene may be expressed from additional subgenomic RNA by including an additional subgenomic RNA promoter on the (-) strand.

[0113] Transgenic plants having one or more trans-acting factors fused to a DNA-dependent RNA polymerase promoter and terminator are obtained. Such factors may include viral replicase (126K / 183K), motor protein (30K), or coat protein. At least one cis-acting sequence required for TMV RNA replication is removed from the transgene. The trans-acting factor may be stably expressed in plant cells, or when an inducible promoter is used, its expression may be induced.

A DNA-dependent RNA polymerase promoter fused exactly to the 5 ′ end of the TMV cDNA,
At least one cis-acting element, such as the 5 'and 3' terminus, important for the TMV life cycle, instead of the trans-acting factor expressed from the chromosome, such as the assembly origin.
A DNA delivery plasmid is constructed that contains two foreign genes or sequences and a ribozyme or convenient restriction enzyme site at the 3 'end. Alternatively, the foreign gene sequence can be expressed from an additional subgenomic RNA promoter, and the sequence encoding the trans-acting factor expressed from the transgene deleted from the DNA delivery plasmid. Preferably, if the viral replicase protein is to be expressed in transgenic plants, the DNA delivery plasmid will have a deletion at nucleotides 3420 to 4902, which appears to be a region that inhibits replication in trans (Lewandowski ( Lewandowski) et al., 1998).

Example 19 Potato Virus X Potato virus X (PVX) has a single-stranded positive-sense RNA genome. Five'
The end has m7Gppp and the 3 'end is polyadenylated. PVX full-length cDNA
Clones were constructed to obtain infectious RNA transcripts (Hemenway et al., 1990).

A DNA delivery plasmid based on PVX RNA containing a PVX cDNA that correctly fuses with a DNA-dependent RNA polymerase promoter at the 5 ′ end and a polyadenylation site at the 3 ′ end is constructed. A convenient restriction enzyme site may be included at the 3 'end. Foreign genes may be expressed from additional subgenomic RNA.

[0117] Transgenic plants are obtained that have one or more trans-acting factors fused to a DNA-dependent RNA polymerase promoter and terminator. Such factors include the viral RNA polymerase gene (ORF 1-147K), the coat protein (ORF 5-21K), or the triple gene block (ORF 2-25K, ORF 3-12K, ORF
4 to 8K). Triple gene block genes can be expressed individually. Alternatively, they can be expressed as minus-sense sense transcripts that can transcribe plus-sense subgenomic RNA for ORFs 2, 3, and 4 by viral replicase. Such a transgene has a DNA-dependent RNA polymerase promoter fused in the minus strand direction with the ORF2, 3 and 4 sequences, and the transcribed sequence is likely to include a subgenomic RNA promoter. PVX
At least one cis-acting sequence required for RNA replication is excised from the transgene. The trans-acting factor may be stably expressed in plant cells or, if an inducible promoter is used, induce its expression.

Transformations expressed from chromosomes, such as the DNA-dependent RNA polymerase promoter fused exactly to the 5 ′ end of the PVX genome, cis-acting elements important for the PVX life cycle such as the 5 ′ and 3 ′ ends, the origin of assembly, etc. A DNA delivery plasmid is constructed that contains at least one foreign gene or sequence in place of the agent, and a polyadenylation signal. Alternatively, the foreign gene sequence can be expressed from an additional subgenomic RNA promoter, and the sequence encoding the transgenic factor that is to be expressed transgenic can be deleted from the DNA delivery plasmid.

Alternatively, a DNA-dependent RNA polymerase promoter, a polyadenylation site, and a DNA having a PVX cDNA sequence in which ORF2 (25K) has been replaced with a foreign gene or sequence.
Construct the NA delivery plasmid. Alternatively, ORF2 is deleted and the foreign gene is expressed from a further subgenomic RNA promoter. Such DNA delivery plasmids
Tobacco mosaic virus (TMV 30K), tomato mosaic virus (ToMV 30K),
Alternatively, transgenic plants expressing motor proteins from a heterologous virus, such as purple clover necrosis mosaic virus (RCNMV 35K), are inoculated.

Example 20-Flok house virus Flok house virus (FHV) has a genome consisting of two single-stranded RNAs. RNA1 encodes protein A involved in RNA replication and encodes protein B translated from sg RNA3 and is not important for RNA replication. RNA2 encodes virion capsid precursor protein α. FHV is used in insect, plant, mammalian, and yeast cells (
Infects Selling et al., 1990; Price et al., 1996).

At its 5 ′ end, it is correctly fused to a DNA-dependent RNA polymerase promoter and its 3 ′
DNA delivery plasmids are constructed for FHV RNA1 and RNA2 containing FHV RNA cDNA fused to a self-cleaving ribozyme at the end. A polyadenylation signal may also be included. Alternatively, a convenient restriction enzyme site may be engineered at the 3 'end. A foreign gene or sequence may be expressed in several ways. For example, a DNA delivery plasmid based on FHV RNA1 can be used to transfer foreign genes or sequences expressed from subgenomic RNA3 to ORF B
It may be included as a substitution or as a translational fusion with ORF B. Alternatively, the foreign gene may be expressed from additional sg RNA. A DNA delivery plasmid based on FHV RNA2 may contain a foreign gene or sequence expressed as part of polyprotein α. The foreign gene in such a construct may include sequences necessary for polyprotein cleavage. The DNA delivery plasmid will preferably express a heterologous plant virus motility protein such as 30K for TMV or 35K for RCNMV. Alternatively, a DNA delivery plasmid would be inoculated into a transgenic plant expressing such a motility protein.

A transgenic plant is obtained that has one or more trans-acting factors fused to a DNA-dependent RNA polymerase promoter and terminator. Such factors may include protein A or the capsid protein precursor α, and will preferably include motility proteins from plant viruses, such as 30K for TMV or 35K for RCNMV. The trans-acting factor may be stably expressed in plant cells or, if an inducible promoter is used, induce its expression. The transgenic expressed trans-acting element preferably lacks at least one cis-acting element required for replication, such as the 5 'and / or 3' end.

A DNA-dependent RNA polymerase promoter fused exactly to the 5 ′ end of RNA1 (or RNA2), at least one cis-acting element important for FHV RNA1 (or RNA2) replication, such as the 5 ′ and 3 ′ ends. A DNA delivery plasmid based on FHV RNA1 or FHV RNA2 containing a foreign gene or sequence and a self-cleaving ribozyme at the 3 ′ end is constructed. A polyadenylation signal may also be included. Alternatively, a convenient restriction enzyme site may be engineered at the 3 'end of the modified viral RNA sequence of the DNA delivery plasmid. A DNA delivery plasmid based on FHV RNA1 may contain a foreign gene or sequence in place of ORFA. Alternatively, ORF A may be deleted and the foreign gene expressed from subgenomic RNA3, for example, as an ORF B substitution or as a translational fusion with ORF B. Alternatively, the DNA delivery plasmid may contain two exogenous RNA sequences, one instead of ORFA and the other expressed from subgenomic RNA3. FHV
An RNA2-based DNA delivery plasmid may contain a foreign gene or sequence in place of ORFα, or may be expressed as part of polyprotein α. The foreign gene in such a construct may include sequences necessary for polyprotein cleavage.

Example 21-Tomato Spotted Wild virus Tomato Spotted Wild virus (TSWV) is divided into three (RNA L, M, S), minus strand sense and antisense (anbisence). ) Is a single-stranded RNA virus.

[0125] 1 fused to a DNA-dependent RNA polymerase promoter and terminator
A transgenic plant having one or more trans-acting factors is obtained.
Such factors include the putative TSWV polymerase gene (ORF L), ORF N, and possibly other trans-acting factors (NSm or NSs). At least one cis-acting sequence, such as the 5 'and / or 3' end, required for TSWV RNA replication is excised from the transgene. The trans-acting factor may be stably expressed in plant cells or, if an inducible promoter is used, induce its expression.

A TSWV RNA M based DNA delivery plasmid is constructed in which the G1 and G2 coding sequences have been replaced by at least one foreign gene or sequence. Such a DNA delivery plasmid contains a DNA-dependent RNA polymerase promoter and a TSWV RNA M cDNA fused to a self-cleaving ribozyme at the 5 'and 3' ends. Alternatively, a DNA delivery plasmid based on TSWV RNA S in which the N coding region has been replaced by a foreign gene or sequence is constructed.

Example 22-Barley Mild Mosaic Virus The barley mild mosaic virus (BaMMV) genome consists of two positive-sense, single-stranded 3 'polyadenylated RNAs. RNA1 is potyvirus P3, 6K1, CI
, 6K2, NIa-VPg, NIa-Pro, NIb, and proteins related to the capsid protein (Kashiwazaki et al., 1990). RNA2 encodes P1 and P2 proteins (Kashiwazaki et al., 1990). P1 protein is potyvirus HC-
Related to Pro, the P2 protein is important for fungal transmission. An isolate containing a defect in the P2 protein was obtained (Timpe and Kuhne, 1995) and
2 is not important for viral RNA replication.

DN for BaMMV RNA1 and RNA2, including the BaMMV RNA cDNA with the DNA-dependent RNA polymerase promoter correctly fused at the 5 ′ end and a polyadenylation site at the 3 ′ end
Construct A delivery plasmid. A foreign gene or sequence may be expressed in several ways. For example, a DNA delivery plasmid based on BaMMV RNA2 may include a foreign gene or sequence expressed as part of a polyprotein that can be cleaved,
Exogenous proteins can be released.

A transgenic plant is obtained that has a BaMMV RNA1 cDNA lacking the 5 ′ and 3 ′ ends fused to a DNA-dependent RNA polymerase promoter and terminator.

A DNA delivery plasmid based on BaMMV (isolated M) RNA2 is constructed. Such a plasmid contains a DNA-dependent RNA polymerase promoter precisely fused to the 5 'end of RNA2, an RNA2 cis-acting replication signal present at the 5' and 3 'ends, a P1 ORF and a P2 ORF.
, Or a foreign gene expressed as part of a P1 / P2 polyprotein that can be cleaved, and the foreign protein can be released.

The contents of all references cited herein are based on the disclosure of the present invention,
To the extent not inconsistent with the teachings and principles, it is incorporated herein by reference.

The examples and embodiments described herein have been set forth for the purpose of illustration, in which light, various modifications or alterations will be suggested to those skilled in the art, which are also within the spirit of the present application. And should be understood to be within the scope.

References

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows a schematic representation of the production of 1a and 2a proteins in host cells.

FIG. 2 shows an example of an Agrobacterium transformation vector containing an expression cassette capable of expressing a 1a and / or 2a BMV protein.

FIG. 3 shows several Agrobacterium vectors created to transform host plant cells (black squares indicate T-DNA border regions).

FIG. 4 shows the general mechanism of BMV RNA3 delivery and replication.

FIG. 5 illustrates a DNA delivery platform that can be used in accordance with the disclosure contained herein. BMV and CCMV designations indicate cis-acting elements.

FIG. 6 illustrates a DNA delivery platform that can be used in accordance with the disclosure contained herein.

FIG. 7 illustrates a DNA delivery platform that can be used in accordance with the disclosure contained herein.

FIG. 8 illustrates a DNA delivery platform that can be used in accordance with the disclosure contained herein.

FIG. 9 shows an Agrobacterium vector for delivering a DNA delivery platform to plant cells (open triangles indicate T-DNA border regions).

FIG. 10 illustrates a DNA delivery platform that can be used in accordance with the disclosure contained herein. Symbols in FIGS. 5 to 10 35S = CaMV35S promoter t = termination / poly A + sequence Rz = ribozyme NOS = NOS promoter OOA = origin of assembly FG = foreign gene

FIG. 11 shows that BMV replication factors support efficient RNA3 replication in protoplasts.

FIG. 12 shows efficient replication of delivered BMV RNA3 in protoplasts.

FIG. 13. Tobacco (N. tabacum) and N. benthamian (N. benthamian)
FIG. 2 shows the transgenic expression of BMV 1a and 2a mRNA in a).

FIG. 14. (1a + 2a) BMV RN delivered in transgenic plants
4 shows efficient replication of A3.

FIG. 15. (1a + 2a) Delivered BMV RN in transgenic plants
Figure 2 shows successful GUS expression from A3.

FIG. 16. Successful GU from delivered BMV RNA3 in protoplasts
S expression is shown.

FIG. 17. (1a + 2a) Delivered BMV RN in transgenic plants
Figure 9 shows successful GFP expression from A3.

FIG. 18. Successful GF from delivered BMV RNA3 in protoplasts
Shows P expression.

FIG. 19 shows efficient replication of delivered BMV RNA3 in (1a + 2a) transgenic N. benthamiana using Agrobacterium inoculation.

FIG. 20 shows (1a + 2a) successful GUS expression from delivered BMV RNA3 with SHMV coat protein in transgenic plants.

FIG. 21 shows that delivered BMV replicates, translocates between cells, and spreads long distances in (1a + 2a) transgenic plants.

FIG. 22: (1a + 2a) Transgenic N. benthami
5 shows transfection of the BMV RNA3 DNA delivery platform into progeny from ana) and localization in the root of the delivered RNA3.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 5/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR) , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU) , TJ, TM), AE, AL, AU, BA, BB, BG, BR, CA, CN, CU, CZ, EE, GD, GE, HR, HU, ID, IL, IN, IS, P, KP, KR, LC, LK, LR, LT, LV, MG, MK, MN, MX, NO, NZ, PL, RO, SG, SI, SK, SL, TR, TT, UA, UZ, VN , YU, ZA (72) Inventor German Thomas El. Orandale Sandy Rock Road 1671, Wisconsin, United States of America 1671 (72) Inventor Arkist Paul G. 3106 Madison Bluff Street, Wisconsin, United States

Claims (30)

[Claims] 1. A DNA-launching platform comprising: a) a polynucleotide molecule encoding a modified viral RNA molecule; and b) a DNA-dependent RNA polymerase promoter. 2. The DNA delivery platform of claim 1, further comprising a sequence encoding at least one cis-acting element. 3. The DNA delivery platform of claim 1, further comprising a ribozyme sequence. 4. The DNA delivery platform of claim 1, further comprising a termination sequence. 5. The DNA delivery platform of claim 1, further comprising a restriction enzyme site. 6. The method of claim 1, wherein the modified RNA molecule comprises an exogenous RNA segment.
The described DNA delivery platform. 7. The DNA delivery platform according to claim 1, wherein the DNA-dependent RNA polymerase promoter is capable of functioning in a plant cell. 8. A method for modifying the genotype or phenotype of one or more cells, comprising the steps of: a) obtaining a DNA delivery platform comprising a polynucleotide molecule encoding a modified viral RNA. And b) transfecting one or more cells with the DNA delivery platform, wherein the polynucleotide molecule is transcribed, thereby producing a replicable RNA transcript. 9. The method of claim 8, further comprising the step of pre-transforming cells having at least one polynucleotide molecule encoding at least one trans-acting factor. 10. The method of claim 8, further comprising introducing a trans-acting factor.
The described method. 11. The method of claim 10, wherein introducing the trans-acting factor comprises co-transfection of an expression plasmid comprising a nucleotide sequence encoding the trans-acting factor. 12. The method of claim 10, wherein introducing the trans-acting factor comprises co-transfection of an RNA transcript encoding the trans-acting factor. 13. The method of claim 10, wherein the trans-acting factor is stably expressed. 14. The method of claim 8, wherein the modified viral RNA comprises an exogenous RNA segment. 15. The method of claim 8, wherein the DNA delivery platform comprises a ribozyme sequence. 16. The method of claim 8, wherein said DNA delivery platform comprises a promoter. 17. The method of claim 8, wherein the DNA delivery platform comprises a termination sequence. 18. The method of claim 8, wherein the DNA delivery platform comprises a restriction enzyme site. 19. A cell produced and modified by the method according to claim 8. 20. A method for producing a plant or plant tissue comprising at least one cell with an altered genotype or phenotype, comprising transfecting a cell of the plant or plant tissue with a DNA delivery platform. The method wherein the DNA delivery platform comprises the polynucleotide encoding an RNA molecule that has been modified to transcribe a polynucleotide molecule to produce a replicable RNA transcript. 21. The modified RNA molecule comprises an exogenous RNA segment.
Method according to 20. 22. The method of claim 20, wherein said DNA delivery platform comprises a ribozyme sequence. 23. The DNA delivery platform comprises a promoter.
Method according to 0. 24. The method of claim 20, wherein the DNA delivery platform comprises a termination sequence. 25. The DNA delivery platform comprises a restriction enzyme site.
Method according to 0. 26. Obtaining at least one modified cell produced by the method of claim 8, and subjecting said modified cell to conditions whereby a plant is regenerated therefrom. A method for producing a plant whose genotype or phenotype has been modified, comprising: 27. A plant produced by the method of claim 26. 28. A plant that is a progeny of the plant of claim 27. 29. The method of claim 20, wherein the plant or plant tissue comprises one or more cells transformed with a polynucleotide molecule encoding at least one trans-acting factor and expresses the polynucleotide molecule. Method. 30. The modified viral RNA molecule is capable of replicating only in one or more cells transformed by a polynucleotide molecule encoding at least one trans-acting factor. the method of.